The present invention relates to a method for determining 5-hydroxycreatinine, which method is useful as a test for renal function disorders, uremia, chronic nephritis, and closure of the urinary tract, etc.
5-Hydroxycreatinine has been found to be an intermediate in the production of methylguanidine, which is a primary urinary toxin accumulated in blood of patients suffering from renal failure. It has been clarified that 5-hydroxycreatinine is produced by a nonenzymatic oxidation of creatinine. Using conventional determination methods, 5-hydroxycreatinine is not detected in the sera of healthy persons, but it is detected in the sera of renal failure patients starting in the initial stage of renal failure and increasing as the symptoms worsen. Therefore, 5-hydroxycreatinine has drawn attention as a marker for renal function disorder and its determination is considered to be an important tool for the evaluation of the disease condition and for early diagnosis of patients suffering from renal failure.
It is believed that an hydroxy radical having a very high reactivity participates in the nonenzymatic oxidation of creatinine. Accordingly, 5-hydroxycreatinine, an intermediate in the reaction of conversion of creatinine to methylguanidine, gets much attention not only as a precursor of methylguanidine, a urinary toxin, but also as an index for the production of hydroxy radical in vivo (an index for oxidative stress state). It has therefore been suggested at present that the determination of 5-hydroxycreatinine is useful as a marker for renal function disorders such as renal failure, diabetic nephropathy and nephritis and also as a marker for systemic oxidative stress.
Known methods for determining 5-hydroxycreatinine as a marker for renal disorder include a method using a high performance liquid chromatography (HPLC) by Nakamura, one of the present inventors (Japanese Patent Laid-Open Nos. 04/161854 and 05/119038). In this method, 5-hydroxycreatinine is separated from blood, serum or urine by a high performance liquid chromatography and the resulting 5-hydroxycreatinine is hydrolyzed and converted to methylguanidine. Methylguanidine is then subject to a quantitative determination by means of fluorescent labeling.
In the method described in the above-mentioned laid-open patent gazettes, separation of 5-hydroxycreatinine from blood, serum or urine is carried out using a column of cation-exchange resin which method requires the application of five kinds of separation solvents, in accordance with the method of Higashidate, et al. (Bunseki Kagaku, 33, pages 366-370 (1984)). In this method, the first separation solvent (sodium citrate/hydrochloric acid; pH 3.00) is eluted for 4.5 minutes; the second separation solvent (sodium citrate/hydrochloric acid; pH 3.50) is eluted for 2.8 minutes; the third separation solvent (sodium citrate/hydrochloric acid; pH 5.25) is eluted for 2.4 minutes; the fourth separation solvent (sodium citrate/boric acid/sodium hydroxide; pH 10.00) is eluted for 2.3 minutes; and the fifth separation solvent (1M sodium hydroxide) is eluted for 30 minutes.
The separating method described in the above-mentioned laid-open patent gazettes correlates with the Higashidate et al method, so it is necessary to prepare as many as five types of separation solvents prior to performing a determination. A complex determination process results which requires much labor, such as programming for switching the separation solvents and for control of eluting time. Moreover, the sensitivity of the determination is 0.5-1 nmol/mL (6.5-13 xcexcg/dL) which is not sufficient for the measurement of changes in small amounts of 5-hydroxycreatinine in the samples of living organisms.
Also disclosed in a paper by Nakamura, et al. (Nephron, 66, pages 140-146 (1994)) is a method for separating and determining 5-hydroxycreatinine using a separation solvent prepared by addition of 1 part of dimethyl sulfoxide (DMSO) to 9 parts of 0.4M citric acid to adjust the pH to 5.25. In the method according to the Nakamura, et al. paper, the eluting time is as long as 30 minutes, which is an inefficient method. Further, the sensitivity for the measurement is about 2 xcexcg/dL, which is still insufficient for measuring the changes in small amounts of 5-hydroxycreatinine in or before the initial stage of renal dysfunction. Accordingly, this method is unsatisfactory as well.
In addition, Nakamura et al., in Japanese Patent Publication No. JP 2000 352564, disclose the determination of 5-hydroxycreatinine via the conversion of 5-hydroxycreatinine to methylguanidine prior to fluorescence spectroscopy. The conversion to methylguanidine may be achieved by raising the temperature of a sample containing the 5-hydroxycreatinine in boric acid or in a boric acid solution to hydrolyze the 5-hydroxycreatinine. A ninhydrin method may be used for labeling of the methylguadinine with fluorescence. The pH of the reaction solution obtained by labelling the methylguadinine with fluorescence is adjusted before the high-speed liquid chromatography.
The present inventors have carried out further intensive investigation and have found a method for the rapid determination of 5-hydroxycreatinine with unexpectedly superior sensitivity. The method of the present invention overcomes sensitivity problems, and overcomes the need for a high number of elution solvents and complex control of their elution times.
The present invention relates to a method for determining 5-hydroxycreatinine in a sample, wherein a separation solvent having a pH of about 4.1 to about 4.6 is selected for use in a high performance liquid chromatography (HPLC) step which is carried out with a strongly acidic cation-exchange resin medium. The present invention also comprises a method for separating 5-hydroxycreatinine contained in a sample by subjecting the sample to HPLC in a strongly acidic cation-exchange resin medium using a separation solvent having a pH of about 4.1 to about 4.6.
The present invention provides a highly sensitive and practical method for determining 5-hydroxycreatinine, which method is useful in testing for renal function disorders such as renal failure, diabetic nephropathy, nephritis, uremia, and closure of the urinary tract, and also for systemic oxidative stress. The method of the present invention comprises performing a high performance liquid chromatography to separate 5-hydroxycreatinine from bodily fluids, urine or other materials sampled from animals and from human beings.
Preferred embodiments of the present invention are:
(1) A method for determining 5-hydroxycreatinine comprising performing HPLC in a column or exchange resin medium comprising a strongly acidic cation-exchange resin, wherein a separation solvent having a pH of about 4.1 to about 4.6 is used;
(2) The method for determining 5-hydroxycreatinine according to the above method (1), wherein the strongly acidic cation-exchange resin is a sulfonic acid cation-exchange resin of the styrene-divinylbenzene series;
(3) The method for determining 5-hydroxycreatinine according to any of the above methods (1) or (2), wherein the separation solvent comprises a mixture of sodium citrate and dimethyl sulfoxide;
(4) The method for determining 5-hydroxycreatinine according to one of the above methods (1) to (3), wherein said 5-hydroxycreatinine is determined from bodily fluid or urine derived from animals;
(5) The method for determining 5-hydroxycreatinine according to the above method (4), wherein 5-hydroxycreatinine is determined from bodily fluid or urine derived from a human being;
(6) The method for determining 5-hydroxycreatinine according to the above methods (4) or (5), wherein 5-hydroxycreatinine is determined from blood;
(7) The method for determining 5-hydroxycreatinine according to the above method (6), wherein 5-hydroxycreatinine is determined from serum; and
(8) The method for determining 5-hydroxycreatinine according to the above methods (4) or (5), wherein 5-hydroxycreatinine is determined from urine.
(9) A method for separating 5-hydroxycreatinine comprising subjecting a sample containing 5-hydroxycreatinine to HPLC in a strongly acidic cation-exchange resin medium using a separation solvent having a pH of about 4.1 to about 4.6.